Background/Objective: Epigenetic regulators control gene expression patterns and have been shown to regulate epidermal differentiation. Modifications of chromatin, for example, methylation, are associated with gene repression as well as activation. The Polycomb group (PcG) proteins tri-methylate histone H3 lysine 27 generating a repressive mark on epidermal differentiation genes and maintaining a proliferative state. This action is opposed by the JMJD3 histone demethylase, which removes these tri-methyl marks and activates epidermal gene expression. Progression of epidermal neoplasias are characterized by loss of differentiation. Thus, we propose that the histone demethylase JMJD3 is a critical player in cancer and loss of function of JMJD3 contributes to progression to epidermal squamous cell carcinoma. Conversely, we propose that the PcG protein, EZH2, whose methyltransferase activity maintains a proliferative state and opposes JMJD3 function, is also involved in progression to invasive squamous cell carcinoma. In support of this idea, overexpression of EZH2 has been observed in a number of cancers. Specific Aims and Research Design: (I) Characterization of JMJD3 function in epidermal neoplasia: (lA) Induction of epidermal neoplasia under altered JMJD3 function;(IB) Determine the contribution of JMJD3 subcellular localization to induction of epidermal neoplasia;(IC) Analysis of JMJD3 function in neoplasia. (II) Determination of the role of PcG protein EZH2 in epidermal neoplasia: (IIA) Induction of epidermal neoplasia with functionally altered EZH2;(IIB) EZH2 impacts on progression of epidermal neoplasia. Loss of function of JMJD3 and EZH2 will be obtained by RNA interference and enzyme-deficient mutants;and gain of function will be obtained by introduction of wild-type protein. Both RNAi and protein expression plasmids will be introduced retrovirally into keratinocytes. We will use a Ras-driven epidermal neoplasia model that is well established in our lab to architecturally recapitulate neoplasia in vivo and assess the role of JMJD3 and EZH2 in tumor progression. PUBLIC HEALTH RELEVANCE: Cancer progression is characterized by loss of cellular differentiation. This proposal seeks to understand mechanisms controlling epidermal growth and differentiation. Abnormalities of epidermal homeostasis include diseases exerting negative impacts on a large portion of the US population, such as squamous cell carcinoma. The current proposal is designed to provide insight and identify targets for the development of future therapies for epidermal cancer.